摘要
目的：恶性黑色素瘤（malignant melanoma, MM）是来源于黑色素细胞的恶性肿瘤，多发生于皮肤，占所有皮肤恶性肿瘤的第三位。其恶性程度高，早期易发生淋巴和血道转移，愈后差。手术切除、生物治疗、放射治疗及化学治疗是恶性黑色素瘤通常采用的治疗方法，但效果均不理想。随着早期诊断、外科手术及辅助治疗的发展，早期患者通过手术多可以治愈，5年生存率约95%，但进展期患者中生存期仅2-8个月，5年生存率不足5%。传统化疗对恶性黑色素瘤的疗效较差，因此，寻求疗效好、毒副作用小的新的治疗方法和药物是肿瘤学者研究的热点问题。
维甲酸类（retinoid）是一组包括维生素A的天然及人工合成的衍生物，具有抗增殖及诱导分化等作用，对多种肿瘤细胞具有抑制作用，是一种有效预防皮肤肿瘤的药物。其抗皮肤黑色素瘤的机制尚未完全阐明。TNF-a具有广泛的生物学活性，具有强大的抗肿瘤作用，是迄今发现的抗肿瘤作用最强的细胞因子，但其副作用较大。二者联合应用，有可能提高肿瘤对诱导剂的敏感性，增强抗肿瘤作用，从而降低单药剂量，减少毒副作用，也可提高治疗效果。本研究旨以人黑色素瘤细胞株A375为研究对象，探讨维甲酸、TNF-a在不同浓度、不同作用时间对皮肤黑色素瘤细胞增殖抑制作用及形态分化的影响，筛选两药联合应用的最佳作用时间和剂量；探讨阿维A酸及两药联合应用对人A375细胞表达MMP-1、TIMP-1的影响。为TNF-a、维甲酸类药物诱导皮肤肿瘤细胞凋亡的基础研究及临床应用提供实验依据。
方法：
1细胞培养将A375恶性黑色素瘤细胞培养于含10%胎牛血清的DMEM培养基（含青霉素100U/ml，含链霉素100U/ml，PH7.2）中，放入37℃、5%CO2的培养箱内进行常规传代培养。
2采用MTT比色法检测阿维A酸及阿维A酸联合TNF-a对A375细胞增殖的抑制作用。
3倒置相差显微镜观察各组药物处理后A375细胞的分化及形态变化。
4应用免疫细胞化学s-p法，检测不同浓度药物处理后A375细胞MMP-1、TIMP-1蛋白的表达状况。
5采用流式细胞分析技术，检测A375细胞周期变化和凋亡。
6采用统计软件SPSS13.0对数据进行统计分析。采用单因素方差分析，以ａ=0.05为显著性差异标准。
结果：
1MTT比色分析法检测各处理因素对人A375细胞增殖的影响（相对抑制率）。
1.1阿维A酸组：浓度不同的阿维A酸组与对照组相比，阿维A酸在0.1umol/L至100umol/L范围内对人黑色素瘤A375细胞有明显的增值抑制作用，且以100umol/L浓度组作用48h抑制率最高。统计分析表明，在12、24及48三个时间段，阿维A酸对人黑色素瘤细胞A375的抑制作用呈现明显的剂量依赖效应关系和时间依赖效应关系，各组间差别具有显著性（p＜0.05）。而10、100μmol/L两组间比较,其抑制作用差异无统计学意义(P>0.05)
1.2阿维A联合TNF-a组：将实验分为3组，分别为阴性对照组（不加任何药物）﹑TNF-a组(5u/L)及阿维A酸(10umol／L)联合TNF-a(5u/L)组。上述各组作用A375细胞48小时后，MTT法检测实验组抑制率TNF组为38.60%，联合组为58.45%，联合组明显高于对照组11.85%和阿维A酸组43.65%，可见阿维A酸和TNF-a联合应用明显抑制A375细胞增殖，与阿维A酸、TNF-a单独应用比较，差异具有显著性（p＜0.05）。
2阿维A酸及阿维A联合TNF-a对A375细胞形态及分化的影响
阴性对照组细胞贴壁生长良好,分布均匀，胞体胞浆饱满呈棱形、梭形或多角形，透明，折光性好，增殖旺盛；阿维A酸处理A375细胞24小时后，细胞变圆、皱缩、破裂，细胞透亮度和折光性减弱，贴壁差，并且出现细胞漂浮等现象，随着作用时间的延长上述现象更加明显；阿维A联合TNF-a作用细胞24小时后，细胞形态变化较单用阿维A更明显，细胞数目明显减少，显微镜下可见多量漂浮细胞。
3免疫细胞化学法检测阿维A酸及阿维A酸联合TNF-a作用前后A375细胞MMP-1、TIMP-1蛋白表达情况
MMP-1、TIMP-1蛋白主要表达于细胞浆内，DAB染色为棕黄或棕褐色。经不同浓度药物处理后的细胞蛋白阳性表达的程度不同，阴性对照组细胞MMP-1高表达，TIMP-1低表达，在阿维A组及联合用药组A375细胞中，MMP-1及TIMP-1蛋白表达均降低，且联合组MMP-1及TIMP-1蛋白表达降低更明显，差异具有显著性（p＜0.05）。
4流式细胞术检测不同浓度药物处理后细胞的周期、凋亡变化
流式细胞术检测显示：与对照组相比（28.29%），加药组（TNF组为38.35%、阿维A酸组为49.56%及联合组为58.62%）中处于G0／G1期的细胞百比例明显增加，而S期（对照组为54.8%、TNF组为42.71%、阿维A酸组为30.5%及联合组为22.03%）的细胞数量明显减少，各组药物均能使G1期延滞，G1-S期转化抑制，将细胞阻滞于G0／G1期，且联合组效果更明显。可见，阿维A联合TNF-a应用对细胞周期的影响具有协同作用。TNF-a及阿维A联合TNF-a均能诱导A375细胞凋亡，但阿维A联合TNF-a诱导A375细胞凋亡作用显著，差异具有显著性（p＜0.05）。
结论：
1阿维A酸和TNF-a都能抑制A375细胞的增殖，并且在一定的浓度和时间范围内，阿维A酸对A375细胞增殖的抑制率具有时间依赖性和量效依赖性。
2阿维A联合TNF-a对A375细胞的增殖抑制率优于阿维A和TNF-a单用，二者联合应用具有协同作用。
3TNF-a及阿维A酸联合TNF-a应用均可阻断人黑色素瘤细胞A375从G0/G1期进入S期，且联合用药诱导A375细胞凋亡的作用更明显
4阿维A酸联合TNF-a对A375细胞的抑制作用可能与下调MMP-1、TIMP-1的表达致使MMPs/TIMPs平衡失调有关，这可能为其抗肿瘤机理有关。
Objective：Malignant melanoma is derived from the melanin cell ofmalignant tumor, occurs more often in the skin, accounting for all skin ofmalignant tumor third.Its highiy malignant and prone to early lymphatic andhematogenous metastasis, the prognosis poor. Surgical resection, biologicaltreatment, radiotherapy and chemotherapy are commonly used treatment ofmalignant melanoma, but the results are not satisfactory. With early diagnosisand surgical intervention and the development of the auxiliary treatment,patients with early by more than surgery can be cured,5years of survival rateof about95%, but in patients with adanced surial only2to8months,5yearsof survival rate to less than5%. Traditional chemotherapy for malignantmelanoma curative effect is poorer, therefore, seeks good effect and sideeffects of small new treatment and drugs are hotspot oncolosts.
Retinoids includes vitamin A, natural and synthetic derivatives. It has theantiproliferation and inducing differentiation effects on a variety of tumor cells.It is also a kind of effective prevention of skin cancer drugs, but the resistanceof skin melanoma mechanism has not been fully elucidated. TNF-a has a widerang of biological activity, with a strong anti-tumor effect, is found so fa, thestrongest anti-tumor effect of cytokines, but its side effects. They mayimprove the sensitivity of the tumor indecer, enhanced anti-tumor effect,which erduces the signle drug dose, and to erduce the toxic side effects, butalso can improve treatment. The aim of this study to the human melanoma cellline A375to investigate retinoid, TNF-a in different concentrations anddifferent time of skin melanoma cells inhibit the proliferation andmorphological differentiation of the screening of the two drugs combined bestthe role of time and dose.Explore the acitretin, and the combination of twodrug to the expression of MMP-1and TIMP-1in human A375cells.TNF-a, Retinoids for skin tumor cell apoptosis in basic research and clinicalapplication provide an experimental basis.
Method:
1Cell culture
The human melanoma cell line A375were cultured in DMEM mediumsupplemented with10%heat inactivated fetal bovine serum,100u/mlpenicillin and100u/ml streptomycin and in a numidified atmosphere5%CO2at37℃.
2Detection of cell growth inhibition with the MTT analysis method.
3The morphological and differentiation changes of cells were detected by thereverse microscopy after the treatment of different drugs.
4To detect the expression of MMP-1, TIMP-1of the human melanoma cellline A375in the different groups by immunohistochemical method.
5Statistic analyses: Data were analyzed by statistical software Spss13.0forwindows. Based on the One-way ANOVA with a=0.05as significant differences standards.
Results:
1To detect growth inhibition rate of A375cells by MTT:
1.1In group acitretin: The difference of inhibition rate among the three groupswas significant(p＜0.05).The results of MTT showed that acitretin inhibitedthe proliferation of A375cells ina dose-dependent and time-dependentmanner.
1.2In group combination of acitretin and TNF-a: The A375cells were dividedin there groups and treated with acitretin in combination or not with TNF-a invitro. The results showed that the combination of acitretin with TNF-ainhibited growth of A375cells, inhibitory rate of combination has remarkabledifference compared with acitretin and control group. The effect of acitretinand TNF-a were synergistic.
2To observe the differentiation and morphology of A375cell by differentdrugs.
Cells in the negative control group grow well stick wall, uniform distribution, moderately sized, cytoplasm full, most in shuttle or polygon shap,transparent, refractive index of proliferation exuberant. Cells go round, shrinks,rupture after treatment by acitretin after24hours. Refraction weakened andfloating cells appeared, along with the role the extension of time of the abovephenpmenon more apparent. The cells treated by acitretin and TNF-a werepresent in the supernatant. The density of adherence cells was diminishedwhen compared with the control.
3To detect the exepression of MMP-1, TIMP-1protein by cellimmunochemistry.
MMP-1, TIMP-1protein was expressed in the intracytoplasm of A375cell. We can detect the expression of MMP-1, TIMP-1protein diminished inarsenic acitretin-treated cells compared with the untreated controls. MMP-1,TIMP-1protein in A375cells treated with acitretin/TNF-a was diminishedsignificantly compared with control and acitretin, TNF-a alone. There hasdifference between the control and the exeperiment groups (p<0.05).
4To detect the cell apoptosis rate and the cell cycle distribution by flowcytometry machine (FCM):
Compared with the control group, medication in each group couldincreased G1-phase population and reduced the S-phase population in A375cells. Application of acitretin and TNF-a combined has synergistic effect;However, TNF-a and combination therapy could induce the apoptosis of A375cells. The combination therapy-induced apoptosis of A375cells moresignificant.
Conclusion:
1Acitretin and TNF-a could significantly inhibit the proliferation of A375cells. All these effects were in dose-dependent and time-dependent mannerwithin the appropriate extent.
2The synergistic effects of a combination of acitretin with TNF-a on inducingdifferentiation, apoptosis and growth inhibition of A375cells which is moreoutstanding than used alone significantly.
3The experiment groups all could increase the proportion of G0/G1period and decrease the proportion of S period, and the apoptosis of A375cells wasmore observed. But combination therapy-induced apoptosis of A375cells ismore obvious.
4The combination of acitretin and TNF-a can inhibit the proliferation of A375cell, which may relate to decreasing the expression of MMP-1, TIMP-1.Thismay be related to its anti-tumor mechanism.
引文
1Vivat Hannah V, Zusi FC. Retinoids as therapeutic agents: today andtomorrow. Mini Rev Med Chemo,2005;5(8):755-760
2Niles RM. Recent advances in the use of vitamion A (Retinoids)in theprevention and treatment of cancer. N Μtrition,2000;16(11):1084-1089
3Baroni A, Paoletti I, Silvestri I,et al.Early vitronectin receptordownregulation in a melanoma cell line during all-trans retinoicacid-induced apoptosis［J］. Br J Dermatol,2003,148(3):424-33
4Zhang H, Satyamoorthy K, Herlyn M,et al. All-trans retinoic acid(atRA)differentially induces apoptosis in matched primary and metastaticmelanoma cells-a speculation on damage effect of atRA via mitochondrialdysfunction and cell cycle redistribution ［J］.Carcinoge-nesis,2003,24(2):185-91
5Njar VC, Gediya L, Purushottamachar P,et al. Retinoic acid metabolismblocking agents(RAMBAs) for treatment of cancer and dermatologicaldisease. Bioorg Med Chem,2006;14(13):4323-4340
6Smith MA,Parkinson DR,Cheson BD,et al.Retinoids in cancer therapy.[J]JClin Oncol.1992May;10(5):839-864
7Baroni A, Paoletti I, Silvstri I, et al.Early vitronectin receptordownregulation in a melanoma a cell line during all-trans retinoicacid-induced apoptosis [J].Br J Dermatol,2003,148(3):424-433
8Lee CS. A review of acitretin, a systemic retinoid for the treatment ofpsoriasis.Expert Opin Pharmacother,2005,6(10):1725-1734
9罗权，张锡宝。阿维A治疗儿童角化性皮肤病的研究状况。国际皮肤性病学杂志，2006,32（3）:136-138
10Otley CC, Stasko T, Tope WD,et al.Chemoprevention of nonmelenomaskin cancer with systemic retinoids:practical dosing and management ofadverse effects.Dermatol Surg,2006,32(4):562-568
11MckeagueAL, wlison DJ, Nelson.Staurosporine-induced apoptosis andhydrogen peroxid-induced necrosis in two human breast cell lines.Br JCancer.2003,88(1):125-131
12Witter LM,Santala SM,Engle L,et al.Decreased response to paclitaxelversus docetaxel in HER-2/neu transfected human breast cancer cells.Am JClin Oncol.2003,26(1):50-54
13Chambers AF,Groom AC,MacDonald IC.Dissemination and growth ofcancer cells in metastatic sites.Nature Rev Caner,2002，2:563-572
14Stelter-StevensonWG,Azanvoorian S，Liotta LA.Tumor cell interactionswith the extracellular matrix during invasion and metastasis. Ann Rev CellBiol,1993,9:541-57
15Charonrat P, Rhys-Evans PH. Eccles SA.Expression of matrixmetalloproteinases and their inhibitors correlates with invasion andmetastasis in squam ous cell carcinoma of the head and neck [J].ArchOtolaryngol Head Neck Surg,2001,127:813-820
16Huntington JT, Shields JM,Der CJ,et al.Overexpression of collagenase1(MMP-1) is mediated by the ERK pathway in invasive melanoma cells:role of BRAF mutation and fibroblast growth factor signaling[J].J BiolChem,2004,279:33168-33176
17Natayi C, Labuousse AL, Debret T,et al.Elastin-derived peptides upregulate matrix metalloproteinase-2-mediated melanoma cell invasionthrough elastin-binding protein[J].J Invest Dermatl,2004,122:256-265
18Huber BF，TheraPeutic oPPOrtunities involving celluolar oneogenes; ovelaPProaches fostered by bioteehnology.FASEBJ,1989;3:5
19Kinoshita T, FukutomiT, Iwamoto E, et a.l Intracystic papillary carcinomaof the breast in a male patient diagnosed bycore needle biopsy: a casereport〔J〕. Breast,2005,14(4):322
20Okita R, Ohsumi S, Takashima S, et a.l Synchronous liver metastases ofintracystic papillary carcinomawith invasion of the breas〔tJ〕.BreastCancer,2005,12(4):327
21Zhang C, Zhang P,Hao J, et a.l High nuclear grade, frequent mitoticactivity, cyclin D1and p53overexpression are associated with stromalinvasion in mammary intracystic papillar carcinoma〔J〕.Breast J,2005,11(1):2
22Grong，QiuMY，Biswas DK.CHa-ras Protooneogene:amplification andoverexPression in induced mouse skin Papillomas and earcinomas.ArehDermatol，2000;126:324
23still，GoldbergLH，Tainsky MA etal.Rasgene mutation and amPlieation inhuman nonmelanoma skin caneers.MolCareinog，2000:4:196
24Baker AH, George SJ, Zaltsman AB, et al. Inhibition of invasion andinduction of apoptotic cell death of cancer cell lines by overexpression ofTIMP-3[J].Br J Cancer,1999,79(9-10):1347-1355
25Nil M, Kayada Y, Yoshiga K, et al. Suppression of metastasis by tissueinhibitor of metalloproteinase-1in a newly established human oralsquamous cell carcinoma cell line [J]. Int J Oncol,2000,16(1):119-120
26马泽粦,樊翌明,吴志华,等.皮肤鳞状细胞癌和基底细胞上皮瘤膜型基质金属蛋白酶-1、基质金属蛋白-2表达[J].中华皮肤科杂志,2004,37:584-586
1Tsao H, Atkins MB, Sober AJ. Management of cutaneous melanoma. NEngl J Med,2004;351(10):998-1012
28Emal A, Murray T, Samuels A, et al. Cancer statistics,2003. CA Cancer JClin,2003;53(1):5-26
29FU SSM,LOREDO L N, BLACHARSKIP A,et al.Proton radiation therapyfor medium and large choroidal melanoma preservation of the eye and itsfunctionality J Int J Radiat OncolBiol Phys,2001,49(4):105321059
30mith RA, Baglioni C. The active form of tumor necrosis factor is atrimmer.J Bil Chem,1987,262:6951
31aroni A, aoletti I, Silvstri I, et al.Early vitronectin receptor downregulationin a melanoma a cell line during all-trains retinoic acid-induced apoptosis[J].Br J Dermatol,2003,148(3):423-433
32MenonC, IyerM, PrabakaranI, et al.TNF-alpha down regulates vascularendothelial Flk-1expression inhuman melanoma a xenograft model.Am JPhysiolHeartCircPhysiol,2003,284:H317
33Carswell EA, Old LJ, Kassel RL, et al.An endotoxin-induced serum factorthat causes necrosis of tumors Proc Natl Acad Sci USA,1975,72(9):3666
34Poehlein CH, HuHM, YamadaJ, etal.TNF plays an essential role in tumorregression after adoptive transfer of perforin/IFN-gamma double knockouteffector T cells. J Immunol,2003,170:2004-2013
35陆琰君,曹纬,等.肿瘤坏死因子在肿瘤研究和治疗领域中的新进展。中国肿瘤生物治疗杂志，2002,9（4）：294-299
36Wang Y, Singh R, Lefk owitch JH,et al.tumor necrosis factor induced toxicliver injury results from JNK2-dependent activartion of caspase8and themitochondrial death path[j].J-Bio Chem.2006,281(22):15258-15267
37杨淑英,段芳龄,潘卫,等. H LA2B7可诱导启动子调控T N F2a和IFNβ基因协同增强抗肿瘤效应[J].中华微生物学和免疫学杂志。2003,23（4）：275-278
38陈震,刘鲁明.重组人肿瘤坏死因子治疗87例中晚期恶性肿瘤.中国肿瘤临床，2003,30:276-279
39HusseinA.N,AIWadei et al.Cyclie AMP-dependent cell type-specifiemodulation of mitogenie signaling by retinoids in normal and neoplasticlung cells.CaneerDeteetPrev.2006，30(5):403-411
40Wu N，ZhangW，YangY，et al.Profilin1obtained by proteomic analysis inall-trans retinoic acid-treated hepatoearcinoma cell lines is involved ininhibition of cell proliferation and migration.Proteomies.2006，6(22):6095-6106
1范玉晶,韩明子.基质金属蛋白酶和消化道肿瘤的相关性[J].世界华人消化杂志,2004,12(9):2174-217
2Risinger GM Jr, Hunt TS, Updike DL, et al. Matrix metalloproteinase-2expression by vascular smooth muscle cells is mediated by bothstimulatory and inhibitory signals in response to growth factors [J]. J BiolChem,2006,281(36):25915-25925
3Visse R, Hideaki N. Matrix metalloproteinases and tissue inhibitors ofmetalloproteinases structure, function, and biochemistry [J]. Circ Res,2003,92:827
4Stewart D, Javadi M, Chambers M, et al. Interleukin-4inhibition ofinterleukin-1-induced expression of matrix metalloproteinase-3(MMP-3)is independent of lipoxygenase and PPARgamma activation in humangingival fibroblasts [J].BMC Mol Biol,2007,8:12
5Preston A. Matrix metalloproteinase [J].Cal Biochem,2002,2:5
6Maskos K, Bode W. Structural basis of matrix metalloproteinase and tissueinhibitors of metalloproteinase [J]. MolBiote Fig20The DNA histogram ofA375cells in5000u/L TNF-a group chnol,2003,25(3):241-266
7Fang J, Shing Y, Wiederschain D, et al. Matrix metalloproteinase-2isrequired for the switch to the angiogenic phenotype in a tumormodel[J].Proc Natl Acad Sci USA,2000,97(8);3884
8Alper O, Bergmann-Leitner ES, Bennett TA,et al.Epidermal growth factorreceptor signaling and the invasive phenotype of ovarian carcinomacells[J].Natl Cancer Inst,2001,93（81）;1375
9Mengshol JA, Vincenti MP, Brinckerhoff CR, IL-1induces collagenase-3(MMP-3) promoter activity in stabiy transfected chondrocytic cells;requirement for Runx-2and activation by p38MAPK and JNK pathways[J].Nucleic Acids Res,2001,29(21);4361
10Murphy G, Docherty JP. The matrix metalloproteinases and their inhibitors.Am J Resp Cell Mol Biol,1992;7(2):120-125
11Hurphy DL, Rudolph LA, Matrisian LM.Matrix metalloproteinases asmediators
12Visse R, Hideaki N·Matrix metalloproteinases and tissue inhibitors ofmetalloproteinases structure, function, and biochemistry [J]·Circ Res,2003,92:827
13Kaija V, Taina TH. Serum tissue inhibitor of metalloproteinase-2(TIMP-2)and matrix metalloproteinase-2in complex with the inhibitor (MMP-2:TIMP-2) as prognostic marker sin bladder cancer [J].Clin Biochem,2007,40(9-10):640-644
14Zhang S, Li L, Lin JY, et al. Imbalance between expression of matrixmetalloproteinases-9and tissue inhibitor of metalloproteinases-1ininvasiveness and metastasis of human gastriccarcinoma [J]. World JGastroenterol,2003,9(5):899-904
15Seo DW, Li H, Guedez L,et al.TIMP-2mediated inhibition of angiogenesis:an MMP-independent mechanism[J]·Cell,2003,114(2):171
16Pang IH, Hellberg PE, Fleenor Dl, et al. Expression of matrixmetalloproteinnases and their inhibitors in human trabecular meshworkcells [J]. Invest Ophthalmol Vis Sci,2003,44(8):3485
17Qi JH, Ebrahem Q, Moor N, et al. A novel function for tissue inhibitor ofmetalloproteinases-3(TIMP-3): inhibition of angiogenesis by blockage ofVEGF binding to VEGF receptor-2[J]. Nat Med,2003,9(4):407
18Docherty AJ, Lyons A, Smith BJ, et al. Sequence of human tissue inhibitorof metalloproteinases and its identity to erythroid-potentiating activity[J].Nature,1985,318(6041);66
19Alper O, Bergmann-Leitner ES, Bennett TA, et al. Epidermal growth factorreceptor signaling and the invasive phenotype of ovarian carcinomacells[J].Natl Cancer Inst,2001,93(18):1375
20曾仲,韩本立.基质金属蛋白酶组织抑制剂的生物学功能[J]·国外医学分子生物学分册,2000,22(2):84
21Majmudar G.Nelson BR.Jensen TC.et al. Increased expression ofstromelysin-3in basal cell carcinomas[J〕.Mol Carcinog.1994.9:17-23.
22Wagener SN.Ruhri C, Kunth K.et al. Expression of stromelysin3in thestromal element of human basal cell carcinoma [J]. Diagn MolPathol.1992.1:200-205
23Thewes M,Worret Wl,Engst R et al. S tromelysin-3(ST-3): immuneo-histochemical characterization of the matrix metaloproteinases (MMP)-11in benign and malignant skin tumors and other skin disorders [J].Clin ExpDermatol.1999.24:122-126
24Wagener SN.Ruhri C. Kunth K.et al. Expression of stromelysin3in thestromal element of human basal cell carcinoma [J]. Diagn MolPathol.1992.1:200-205
25Tsukifuji R.Tagawa K.Hatamoehi A.et al.Expression of matrixmetal-loproteinase-1.-2and-3in squamous cell carcinoma and actinickeratosis [J].Br J Cancer.1999.80:1087-1091
26Luukkaa M.Vihinen P.Kronqvist P.et al.Assoeiation between highcollagenase-3expression levels and poor prognosis in patients with headand neck cancer[J].Head. Nec.2.005.Nov21.「Epub ahead of print]
27Dumas V.Kanitakis J.Charvat S.Euvrard S.Faure M.Claudy A.Expressionof basement membrane antigens and matrix metalloproteinases2and9incutaneous basal and squamous cell careinomas[J].Anticancer Res.1999.19:2929-2938
28Hofmann UB, Westphal JR, Van Kraats AA, et al. Expression of integrinalpha(v)beta(3)correlates with activation of membrane-type matrixMetalloproteinases-1(MT1-MMP) and metalloproteinases-2(MMP2) inHuman melanoma Cells in vitro and in vivo[J]. Int J Cancer,2000,87(1):12-19
29金霞,张健慧.基质金属蛋白酶基因多态性的研究进展[J].国外医学遗传学分册,2005;04(15):28-02
30Nyormoi O, Mills L, Bar-Eli M. An MMP-2/MMP-9inhibitor,5a,enhances apoptosis induced by ligands of the TNF receptor superfamily incancer cells [J]. Cell Death Differ,2003,10(5):558-569
